PROJECT SUMMARY ? TR&D3 Development of protein technologies for use in vivo in the Drosophila system has lagged behind development of molecular genetic technologies. In this project, we will develop protein-based technologies that complement and extend the type of studies that can be done in this exemplary model system. We will focus on development of proximity labeling, as facilitated by peroxidases or biotin ligases, as tools for in vivo proteomics analysis of specific subcellular compartments and signaling. We will additionally develop a robust pipeline for isolation of nanobodies that can be used in diverse applications, including for in vivo expression of fusion proteins that facilitate visualization and functional blocking. Collaborators with interests in using the technologies to study cell biology, development, transcription control, and neuronal networks provide appropriate assays for iterative testing and improvement. Altogether, this project will help fill an important gap in the Drosophila toolbox that can help provide a more complete picture of functions at the subcellular, cellular, organ, and whole animal levels that would not be achievable in cell systems or using only molecular genetic tools.